Lipid kinase

ABSTRACT

The invention relates to a novel lipid kinase which is part of the PI3 Kinase family. PI3 Kinases catalyze the addition of phosphate to inositol generating inositol mono, di and triphosphate. Inositol phosphates have been implicated in regulating intracellular signaling cascades resulting in alternations in gene expression which, amongst other effects, can result in cytoskeletal remodeling and modulation of cellular motility. More particularly the invention relates to a novel human PI3 Kinase, p110Δ which interacts with p85, has a broad phosphinositide specificity and is sensitive to the same kinase inhibitors as PI3 Kinase p110A. However, in contrast to previously identified PI3 Kinases which show a ubiquitous pattern of expression, p110Δ is selectively expressed in leucocytes. Importantly, p110Δ shows enhanced expression in most melanomas tested and therefore may play a crucial role in regulating the metastatic property exhibited by melanomas. The identification of agents that enhance or reduce p110Δ activity may therefore prevent cancer metastatis.

The invention relates to a novel lipid kinase which is part of the PI3Kinase (P13K) family and more specifically the invention relates tovarious aspects of the novel lipid kinase particularly, but notexclusively, to an identification of expression of said kinase with aview to diagnosing or predicting motility or invasion of cells such asmetastasis of cancer cells; and also agents for interfering with saidexpression or inhibiting said kinase with a view to enhancing orreducing or preventing said motility or invasion so as to enhance orrestrict, respectively the movement of selected cells.

An overview of the PI3 kinase family of enzymes is given in ourco-pending Patent Application WO93/21328. Briefly, this class of enzymesshows phosphoinositide (hereinafter referred to after as PI) 3-kinaseactivity. Following major advances in our knowledge of cell signaltransduction and cell second messenger systems it is known that thePI3Ks have a major role to play in regulating cell function. Indeed, itis known that PI3Ks are members of a growing number of potentialsignalling proteins which associate with protein-tyrosine kinasesactivated either by ligand stimulation or as a consequence of celltransformation. Once thus associated they provide an important complexin the cell signalling pathway and thus direct events towards a givenconclusion.

PI3 kinases catalyse the addition of phosphate to the 3′-OH position ofthe inositol ring of inositol lipids generating phosphatidyl inositolmonophosphate, phosphatidyl inositol diphosphate and phosphatidylinositol triphosphate (Whitman et al, 1988, Stephens et al 1989 and1991). A family of PI3 kinase enzymes has now been identified inorganisms as diverse as plants, slime molds, yeast, fruit flies andmammals (Zvelebil et al, 1996).

It is conceivable that different PI3 kinases are responsible for thegeneration of the different 3′-phosphorylated inositol lipids in vivo.Three classes of PI3 kinase can be discriminated on the basis of theirin vitro lipid substrates specificity. Enzymes of a first class have abroad substrate specificity and phosphorylate PtdIns, PtdIns(4)P andPtdIns(4,5)P₂. Class I PI3 kinases include mammalian p110α, p110β andp110γ (Hiles et al, 1192; Hu et al, 1993; Stephens et al, 1994; Stoyanovet al, 1995).

P110α and p110β are closely related PI3 kinases which interact with p85adaptor proteins and with GTP-bound Ras.

Two 85 kDa subunits, p85α and p85β, have been cloned (Otsu et al, 1992).These molecules contain an N-terminal src homology-3 (SH3) domain, abreakpoint cluster (bcr) homology region flanked by two proline-richregions and two src homology-2 (SH2) domains. Shortened p85 proteins,generated by alternative splicing from the p85α gene or encoded by genesdifferent from those of p85α/β, all lack the SH3 domain and the bcrregion, which seem to be replaced by a unique short N-terminus (Pons etal, 1995; Inukai et al, 1996; Antonetti et al, 1996). The SH2 domains,present in all p85 molecules, provide the heterodimeric p85/p110 PI3Kswith the capacity to interact with phosphorylated tyrosine residues on avariety of receptors and other cellular proteins. In contrast to p110αand β, p110γ does not interact with p85 but instead associates with ap101 adaptor protein (Stephens et al, 1996). P110γ activity isstimulated by G-protein subunits.

PI3Ks of a second class contains enzymes which, at least in vitro,phosphorylate PtdIns and PtdIns(4)P but not PtdIns(4, 5)P₂ (MacDougallet al, 1995; Virbasius et al, 1996, Molz et al, 1996). These PI3Ks allcontain a C2 domain at their C-terminus. The in vivo role of these classII PI3Ks is unknown.

A third class of PI3K has a substrate specificity restricted to PtdIns.These PI3Ks are homologous to yeast Vps34p which is involved intrafficking of newly formed proteins from the Golgi apparatus to thevacuole in yeast, the equivalent of the mammalian lysosome (Stack et al,1995). Both yeast and mammalian Vps34p occur in a complex with Vps15p, a150 kDa protein serine/threonine kinase (Stack et al, 1995; Volinia etal, 1995; Panaretou et al, submitted for publication).

PtdIns(3)P is constitutively present in cells and its levels are largelyunaltered upon extracellular stimulation. In contrast, PtdIns(3, 4)P₂and PtdIns(3, 4, 5)P₃ are almost absent in quiescent cells but areproduced rapidly upon stimulation by a variety of growth factors,suggesting a likely function as second messengers (Stephens et al,1993). The role of PI3Ks and their phosphorylated lipids in cellularphysiology is just beginning to be understood. These lipids may fulfilla dual role: apart from exerting physical, charge-mediated effects onthe curvature of the lipid bilayer, they also have the capacity tointeract with specific binding proteins and modulate their localisationand/or activity. Amongst the potential targets for these lipids areprotein kinases such as protein kinase C isoforms, protein kinaseN/Rho-activated kinases and Akt/RAC/protein kinase B (Toker et al, 1994;Palmer et al, 1995; Burgering and Coffer, 1995; Franke et al, 1995;James et al, 1996; Klippel et al, 1996). Akt/RAC/protein kinase B islikely to be upstream of targets such as p70 S6 kinase and glycogensynthase kinase-3 (Chung et al, 1994; Cross et al, 1995). PI3Ks alsoaffect the activity of small GTP-binding proteins such as Rac and Rab5,possibly by regulating nucleotide exchange (Hawkins et al, 1995; Li etal, 1996). Ultimately, the combination of these actions can result incytoskeletal rearrangements, DNA synthesis/mitogenesis, cell survivaland differentiation (Vanhaesebroeck et al, 1996).

We describe herein a mammalian novel Class I PI3 Kinase which we havetermed p110δ. This novel P13 Kinase typifies the Class I PI3 Kinasefamily in that it binds p85α, p85β and p85γ. In addition, it also bindsGTP-ras but, like p110α, shows no binding of rho and rac. It also sharesthe same GTP-broad phosphoinositide lipid substrate specificity of p110αand p110β, and it also shows protein kinase activity and has a similardrug sensitivity to p110α.

However, it is characterised by its selective tissue distribution. Incontrast to p110α and p110β which seem to be ubiquitously expressed,p110δ expression is particularly high in white blood cell populationsi.e. spleen, thymus and especially peripheral blood leucocytes. Inaddition to this observation we have also found that p110δ is expressedin most melanomas, but not in any melanocytes, the normal cellcounterpart of melanomas. Given the natural distribution of p110 intissues which are known to exhibit motility or invasion and also theexpression of p110δ in cancer cells we consider that p110δ has a role toplay in cell motility or invasion and thus the expression of this lipidkinase in cancer cells can explain the metastatic behaviour of cancercells.

A further novel feature of p110δ is its ability to autophosphorylate ina Mn²+-dependent manner. Indeed, we have shown that autophosphorylationtends to hinder the lipid kinase activity of the protein. In addition,p110δ contains distinct potential protein:protein interaction modulesincluding a proline-rich region (see FIG. 1, position 292-311, wherein 8out of 20 amino acids are proline) and a basic region leucine zipper(bZIP) like domain (Ing et al., 1994 and Hirai et al., 1996). Suchbiochemical and structural differences between p85-binding PI3 kinasesindicate that they may fulfill distinct functional roles and/or bedifferentially regulated in vivo.

We disclose herein a nucleic acid molecule, of human origin, andcorresponding amino acid sequence data relating to p110δ. Using thisinformation it is possible to determine the expression of p110δ invarious tissue types and in particular to determine the expression ofsame in cancer tissue with a view to diagnosing the motility orinvasiveness of such tissue and thus predicting the potential forsecondary tumours occurring. Moreover, it will also be possible toprovide agents which impair the expression of p110δ or alternativelyinterfere with the functioning of same. For example, having regard tothe sequence data provided herein it is possible to provide antisensematerial which prevents the expression of p110δ.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding a PI3Kδprotein, to decrease transcription and/or translation of PI3Kδ genes.This is desirable in virtually any medical condition wherein a reductionin PI3Kδ gene product expression is desirable, including to reduce anyaspect of a tumor cell phenotype attributable to PI3Kδ gene expression.Antisense molecules, in this manner, can be used to slow down or arrestsuch aspects of a tumor cell phenotype.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligoneucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene. Those skilled in the art willrecognize that the exact length of the antisense oligonucleotide and itsdegree of complementarity with its target will depend upon the specifictarget selected, including the sequence of the target and the particularbases which comprise that sequence. It is preferred that the antisenseoligonucleotide be constructed and arranged so as to bind selectivelywith the target under physiological conditions, i.e., to hybridizesubstantially more to the target sequence than to any other sequence inthe target cell under physiological conditions. Based upon the DNAsequence presented in FIG. 9 or upon allelic or homologous genomicand/or DNA sequences, one of skill in the art can easily choose andsynthesize any of a number of appropriate antisense molecules for use inaccordance with the present invention. In order to be sufficientlyselective and potent for inhibition, such antisense oligonucleotidesshould comprise at least 7 (Wagner et al., Nature Biotechnology14:840-844, 1996) and. more preferably, at least 15 consecutive baseswhich are complementary to the target. Most preferably, the antisenseoligonucleotides comprise a complementary sequence of 20-30 bases.Although oligonucelotides may be chosen which are antisense to anyregion of the gene or mRNA transcripts, in preferred embodiments theantisense oligonucleotides correspond to N-terminal or 5′ upstream sitessuch as translation initiation, transcription initiation or promotersites. In addition, 3′-untranslated regions may be targeted. Targetingto mRNA splicing sites has also been used in the art but may be lesspreferred if alternative mRNA splicing occurs. In addition, theantisense is targeted, preferably, to sites in which mRNA secondarystructure is not expected (see, e.g., Sainio et al., Cell Mol.Neurobiol. 14(5):439-457. 1994) and at which proteins are not expectedto bind. Finally, although FIG. 9 discloses cDNA sequence, one ofordinary skill in the art may easily derive the genomic DNAcorresponding to the cDNA of FIG. 9. Thus, the present invention alsoprovides for antisense oligonucleotides which are complementary to thegenomic DNA corresponding to FIG. 9. Similarly, antisense to allelic orhomologous DNAs and genomic DNAs are enabled without undueexperimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,phosphate triesters, acetamidates, peptides, and carboxymethyl esters.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. Modifiedoligonucleotides also can include base analogs such as C-5 propynemodified bases (Wagner et al., Nature Biotechnology 14:840-844, 1996).The present invention, thus, contemplates pharmaceutical preparationscontaining modified antisense molecules that are complementary to andhybridizable with, under physiological conditions, nucleic acidsencoding PI3Kδ proteins, together with pharmaceutically acceptablecarriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

It is therefore an object of the invention to identify a novel PI3Kinase and so provide means for predicting the likely motility orinvasiveness of cells.

It is a yet further object of the invention to provide agents thatenhance or reduce or prevent the expression of p110δ and/or agents whichinterfere with the functioning of p110δ, with a view to enhancing orhindering or preventing, respectively, the motility or invasiveness ofcells.

According to a first aspect of the invention there is therefore providedan isolated autophosphorylating polypeptide which possesses PI3 kinaseactivity.

Ideally said polypeptide is derived from white blood cells and istypically expressed in melanomas, more ideally still said polypeptide isof human

Moreover, the polypeptide is capable of association with p85 subunits ofmammalian PI3 Kinases ideally to produce active complexes.

More preferably still the polypeptide has the amino acid sequence shownin FIG. 1A or a sequence homologous thereto which is in particularlycharacterised by a proline rich domain.

Reference herein to the term homologous is intended to cover material ofa similar nature or of common descent or pocessing those features, asherein described, that characterise the protein, or material, whosecorresponding nucleic acid molecule hybridises, such as under stringentconditions, to the nucleic acid molecule shown in FIG. 9. Typicalhybridisation conditions would include 50% formamide, 5×SSPE, 5×Denhardts solution, 0.2% SDS, 200 μg/ml denatured sonicated herringsperm DNA and 200 μg/ml yeast RNA at a temperature of 60° C.,(conditions described in the published patent specification WO93/21328).

Ideally the polypeptide is produced using recombinant technology and istypically of human origin.

According to a further aspect of the invention there is provided anantibody to at least a part of the polypeptide of the invention, whichantibody may be polyclonal or monoclonal.

According to a further aspect of the invention there is provided thewhole or a part of the nucleic acid molecule shown in FIG. 9 whichmolecule encodes an autophosphorylating polypeptide having PI3 Kinaseactivity.

In the instance where said part of said molecule is provided, the partwill be selected having regard to its purpose, for example it may bedesirable to select a part having kinase activity for subsequent use oranother part which is most suitable for antibody production.

According to a further aspect of the invention there is provided anucleic acid molecule construct comprising a whole or a part of thenucleic acid molecule of the invention wherein the latter nucleic acidmolecule is under the control of a control sequence and in appropriatereading frame so as to ensure expression of the corresponding protein.

According to a yet further aspect of the invention there is providedhost cells which have been transformed, ideally using the construct ofthe invention, so as to include a whole or a part of the nucleic acidmolecule shown in FIG. 9 so as to permit expression of a whole, or asignificant part, of the corresponding polypeptide.

Ideally these host cells are eukaryotic cells for example, insect cellssuch as cells from the species Spodoptera frugiperda using thebaculovirus expression system. This expression system is favoured in theinstance where post translation modification is required. If suchmodification is not required a prokaryotic system may be used.

According to a further aspect of the invention there is provided amethod for diagnosing the motility of cells comprising examining asample of said cells for the expression of the polypeptide of theinvention.

Ideally, investigations are undertaken in order to establish whethermRNA corresponding to the polypeptide of the invention is expressed insaid cells, for e.g. by using PCR techniques or Northern Blot analysis.Alternatively, any other conventional technique may be undertaken inorder to identify said expression.

According to a yet further aspect of the invention there is provided amethod for identifying antagonists effective at blocking the activity ofthe polypeptide of the invention which comprises screening candidatemolecules for such activity using the polypeptide, or fragments thereofthe invention.

Ideally, screening may involve artificial techniques such ascomputer-aided techniques or conventional laboratory techniques.

Ideally, the above method is undertaken by exposing cells known toexpress the polypeptide of the invention, either naturally or by virtueof transfection, to the appropriate antagonist and then monitoring themotility of same.

Alternatively, the method of the invention may involve competitivebinding assays in order to identify agents that selectively and ideallyirreversibly bind to the polypeptide of the invention.

According to a yet further aspect of the invention there is provided apharmaceutical or veterinary composition comprising an agent effectiveat enhancing or blocking the activity or expression of the polypeptideof the invention which has been formulated for pharmaceutical orveterinary use and which optionally also includes a dilutant, carrier orexcipient and/or is in unit dosage form.

According to a yet further aspect of the invention there is provided amethod for controlling the motility of cells comprising exposing apopulation of said cells to either an agonist or antagonist or thepolypeptide of the invention or to antisense material ashereindescribed.

Alternatively, in the aforementioned method said cells may be exposedalternatively or additionally, to the polypeptide of the invention witha view to increasing the effective levels of said polypeptide and soenhancing cell motility.

The aforementioned method may be undertaken either in vivo or in vitro.

According to a yet further aspect of the invention there is provided useof an agent effective at blocking the activity of the polypeptide of theinvention for controlling cell motility.

According to a yet further aspect of the invention there is provided useof the polypeptide of the invention for enhancing cell motility.

According to a yet further aspect of the invention there is providedantisense oligonucleotides ideally modified as hereindescribed, forhybridizing to the nucleic acid of the invention.

An embodiment of the invention will now be described by way of exampleonly with reference to the following figures, materials and methodswherein:

FIG. 1(A) shows translated amino acid sequence of human p110δ cDNA. Theproline-rich region and the bZIP-like domain are indicated by open andshaded box, respectively. (B) Dotplot comparison of the full lengthamino acid sequence of p110δ with that of p110α and p110β. Non-conservedsequence motifs are underlined. Dotplot comparisons were performed usingthe COMPARE program (UWGCG package: Devereux et al, 1984). (C)Comparison of the p110δ amino acid sequence flanking HR3 with respectivehomologous regions of p110α and p110β. Amino acid numbering is that ofp110δ. Proline-rich region: critical prolines enabling the formation ofa left-handed polyproline type-II helix in p110δ are indicated with anasterisk bZIP region: conserved L/V/I residues of the leucine-zipperregion are indicated with arrowheads.

FIG. 2. Interaction of p110δ with p85 and Ras (A) Insect cells wereinfected with recombinant baculovirus encoding GST-p110δ, alone or incombination with viruses encoding either p85α, β or γ. After 2 days,GST-p110δ was affinity-purified from the cell lysates usingglutathione-sepharose, washed, and analysed by SDS-PAGE and Coomassiestaining. (B) P110δ was immunoprecipitated from 500 μg human neutrophilcytosol and probed for the presence of different p85 isoforms by Westernblotting. rec=recombinant p85 purified from Sf9 cells. (C) GST-p110α/85αand GST-p110δ/85α (0.25 μg) were incubated with the indicated amount (inμg) of GTP- or GDP-loaded V12-Ras, washed and probed for the presence ofRas by Western blotting as described (Rodriguez-Viciana et al, 1994,1996).

FIG. 3. (A) In vitro lipid substrate specificity of p110δ.GST-p110δ/p85α was used in a lipid kinase assay using the indicatedsubstrates in the presence of Mg²⁺. Equal cpm were spotted at theorigin. (B) HPLC analysis of the PtdIns phosphorylation productgenerated by GST-p110δ/p85α. Elution times of the deacylated product ofp110δ (solid line) and glycerophosphoinositol-3P andglycerophosphoinositol-4P standards (dotted lines) are shown. Thepositions of the AMP and ADP controls are indicated by arrows.

FIG. 4. Protein kinase activity of p110δ. (A) GST-p110α or GST-p110δ, incomplex with the indicated p85 subunits, were subjected to an in vitroprotein kinase reaction in the presence of Mn²⁺, and further analysed bySDS-PAGE, Coomassie staining and autoradiography, (B,C) Untagged p110αand p110δ [wild-type (WT) or kinase defective mutants (p110α-R916P andp110δ-R894P)], in complex with p85α or β on PDGF-receptor phosphopeptidebeads, were subjected to an in vitro kinase reaction and furtheranalysed as described under (A). Open and closed arrowheads point top110 and p85 proteins, respectively. Right panel in (B): phosphoaminoacid analysis of p85α and p110δ.

FIG. 5. Sensitivity of p110δ lipid kinase activity to drugs. Inhibitionof p110δ/p85α (closed circles) and p110α/p85α (open circles) isnormalised to activity in the absence of the drug wortmannin. These datapoints are the mean (±SE) of 3 experiments.

FIG. 6. Northern blot analysis of expression of p110α, p110β and p110δ.

FIG. 7. Analysis of p110α and p110δ protein expression. 100 μg of totalcell lysate was loaded per lane. Platelets were lysed in either lysisbuffer as described under Materials and Methods, or in Laemmli gelloading buffer containing 2-mercaptoethanol. PMBC, peripheral bloodmononuclear cells; PBL, peripheral blood lymphocytes.

FIG. 8. Involvement of p110α and p110δ in cytokine signalling. Ba/F3 (A)and MC/9 (B) cell lines were stimulated with the indicated cytokines.Samples from control untreated cells are labelled Con. Total celllysates, and p110α and p110δ IPs were separated by SDS-PAGE to prepareduplicate blots, the references for which were p110δ/85α (panels a, band d) or p110α/85α (panels c and e). Immunoblotting of native blotswere performed with 4G10 (anti-PTyr, panels a) and anti-p110α (panelsc). Blots were subsequently stripped and reprobed with anti-SHP2. (A,panel b), anti-kit (B, panel b), anti-p110δ (panels d) and anti-p85antibodies (panels e). The arrowheads indicate the positions of p170(IRS-2), p100 and p70 (SHP2) (A, panel a), and of p150 (c-kit) and p100(B, panel b).

FIG. 9. The complete human cDNA sequence of p110δ.

FIG. 10. Represents immunofluorescence images of murine macrophagesmicroinjected with affinity purified antibodies to p110δ. The macrophagecytoskeletons are imaged with phalloidin conjugated rhodamine.

Materials and Methods

Cloning of p110δ

Details of the isolation of partial PI3 kinase cDNA clones via RT-PCRbased on homologous regions between bovine p110α and S. cerevisiaeVps34p have been described (Volinia et al., 1995: MacDougall et al.,1996). This, approach yielded from the MOLT4 T cell line a partial p110δcDNA fragment which was then used to screen an oligo(dT)-primed U937cDNA library (Volinia et al., 1995). Complementary DNA was EcoRI-XhoIcloned in Lambda ZAPII vector digested with EcoRI-XhoI (Stratagene). Outof 4 million clones screened, 6 primary positive plaques were found, 3of which remained positive during two further rounds of screening. ThecDNA inserts in pBluescript were prepared by in vivo excision accordingto the manufacturer's (Stratagene) instructions. Three representativepBluescript clones (0_(5.1), 0_(9.1) and 0_(11.1)) were characterised byrestriction mapping and PCR, and found to contain inserts with sizesranging from 4.4 kb (0_(11.1)) to 5.0 kb 0_(5.1). 0_(9.1)). Clone0_(9.1) was used for detailed characterisation. Restriction mapping ofits insert revealed the absence of an internal XhoI site, and thepresence of 2 internal EcoRI sites, respectively 223 and 3862nucleotides 3′ from the EcoRI cDNA insertion site (nucleotide1=underlined nucleotide of FIG. 9). Consequently, combined EcoRI andXhoI digest divided the 0_(9.1) insert in 3 fragments, further indicatedas EcoRI fragment I (nucleotide 1-222), EcoRI fragment II (nucleotide223-3861) and EcoRI-XhoI fragment III (nucleotide 3862-5000approximately). Both strands of fragments I and IS were sequenced usingthe Taq DyeDeoxy Terminator Cycle sequencing system (ABI) and thecomplete cDNA sequence is shown in FIG. 9. An open reading framespanning nucleotides 195 to 3330 of the 0_(9.1) insert was found. An inframe stop codon precedes the potential start codon, which lies in afavourable context for translation initiation (Kozak, 1991). Thisresults in 196 nucleotides of 5′ untranslated region (UT) andapproximately 2.2 kb 3′ UT. In the sequenced 5′ end of 0_(5.1), 0_(9.1)and 0_(11.1) clones, 2 different but related 5′ untranslated regionswere found indicative for the existence of at least 2 slightly differentmessenger RNAs.

Construction of Expression Vectors for p110δ

Insect cell transfer vectors used were pVL1393 (for untagged p110δ;InVitrogen) and pAcG3X (for GST-p110δ; Davies et al., 1993). The codingregion for p110δ was subcloned in these vectors in two steps. First, theexpression vectors were engineered, via linker insertion at themulticloning site, to contain part of the sequence of EcoRI fragment Iof p110δ, spanning the start codon (at nucleotide 197; see above) to thesecond EcoRI site (nucleotide 223; see above). In the latter EcoRI site,EcoRI fragment II of p110δ was subcloned, followed by selection forclones with correctly orientated inserts. The first step for the insectcell vectors was BamHI-EcoRI cleavage followed by insertion of thefollowing linker (linker I): GATCCCCACCATGCCCCCTGGGGTGGACTGCCCCATGG(sense: 5′-3′) (antisense: 5′-3′) AATTCCATGGGGCAGTCCACCCCAGGGGGCATGGTGGG

This linker contains the ATG with an optimal Kozak consensus sequence(Kozak, 1991). Further derivatives of p110δ were made by PCR using VentDNA polymerase (New England Biolabs). P110δ EcoRI fragment II, subclonedin pBluescript-SK (further indicated as pBluescript-p110δ-EcoII) washereby used as a template. In these PCR reactions, the 3′-untranslatedregion of the EcoRI fragment II insert was removed. Oligonucleotidesused to create the mutation R894P were as follows: sense mutagenicoligonucleotide=PRIMER 1 (mutagenic residue underlined)= sense mutagenicoligonucleotide = PRIMER 1 (mutagenic residue underlined) =5′-GTGTGGCCACATATGTGCTGGGCATTGGCGATCCGCACAGCGACA ACATCATGATCCG,Anti-sense = PRIMER 2 = 5′-GGCCCGGTGCTCGAGAATTCTACTGCCTGTTGTCTTTGGACACGTTGTGGGCC.

A parallel PCR was performed using primer 2, and a sense primer (PRIMER3=5′-GTGTGGCCACATATGTGCTGGGCATTGGCG) leaving the wild type p110δsequence intact. All PCR products were cleaved with NdeI and XhoI,subcloned into NdeI-XhoI-opened pBluescript-p110δ-EcoII and sequenced.Correct clones were then transferred as an EcoRI cassette intoEcoRI-opened pVL1393 containing linker I followed by selection forclones with correctly orientated insert.

Expression of p110δ in Insect Cells

Plasmid DNA was cotransfected with BaculoGold DNA (Pharmingen, SanDiego, Calif.) using Lipofectin reagent (Gibco). Recombinant plaqueswere isolated and characterised by established methods (Summers andSmith 1987).

Cell Culture

Cells were cultured in a humidified 5% CO₂ incubator in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 20 μM 2-mercaptoethanol, 100units/ml penicillin/streptomycin and 2 mM glutamine. Ba/F3 is a murineIL3-dependent pre-B cell line (Palacios and Steinmetz, 1985) and MC/9 isa murine IL3-dependent mast cell line (Nabel et al., 1981). Both Ba/F3and MC/9 were maintained in 10% (v/v) conditioned medium derived fromWEHI3B, as the source of murine IL3. FDMAC11/4.6 (FD-6) myeloidprogenitor cells are an indigenous variant of FDMAC11 which will grow inresponse to IL4, as well as IL3, GM-CSF and CSF-1 (Welham et al.,1994a). These cells were maintained in 3% (v/v) IL4-conditioned mediumderived from the AgX63/OMIL4 cells (Karasuyarna and Melchers, 1988).

Lipid Kinase Assay

Lipid kinase activity was performed essentially as described by Whitmanet al. (1985). Lipid kinase assay buffer was 20 mM Tris HCl pH 7.4, 100mM NaCl and 0.5 mM EGTA. Lipids were purchased from Sigma. The finalconcentration of ATP and Mg²⁺ in the assay were routinely 0.5 and 3.5mM, respectively, while lipids were used at 0.2-0.4 mM concentration.Unless otherwise indicated, kinase reaction was for 10 min at 37° C. Thesolvent for TLC separation of reaction products was propan-1-ol/2 Macetic acid/5 M H₃PO₄ (65:35:1). Assays of drug effects on the kinasewere performed using PtdIns as substrate in the presence of 40 μM ATP(final) for 10 min at 25° C.; all tubes contained 1% DMSO. Activity wasquantified by phosphorimager (Molecular Dynamics) analysis ofTLC-separated lipid products.

HPLC Analysis

[³²P]-PtdIns3P, prepared by phosphorylating PtdIns with recombinantp110α, and [³²P]-PtdIns4P, generated by converting PtdIns with A431membranes in the presence of 0.5% NP-40, were used as standards.Glycerophosphoinositols, generated by deacylation of lipids withmethylamine (Clarke and Dawson, 1981), were separated by anion exchangeHPLC on a PartisphereSAX column (Whatman International) using a lineargradient of 1 M (NH₄)₂HPO₄ against water (0-25% B; 60 min) at 1 ml/min.Radioactive peaks were detected by an on-line detector (ReeveAnalytical, Glasgow). ADP and ATP nucleotide standards, added asinternal controls to ensure consistency between runs, were detected byabsorbance at 254 nm.

In Vitro Protein Phosphorylation Assay and Effect on Lipid KinaseActivity

Precipitated proteins were incubated for 30 min at 37° C. in proteinkinase assay buffer (20 mM Tris.HCl (pH 7.4), 100 mM NaCl, 0.5 mM EGTA,50 μM ATP and 1 mM MnCl₂.4H₂O, 5-10 μCi[γ-³²P]ATP/ml). The reaction wasstopped by addition of SDS-PAGE sample buffer, and the reaction productsanalysed by SDS-PAGE and autoradiography. Phosphoamino acid analysis wasperformed on a Hunter thin layer electrophoresis system (CBS ScientificCo, Del Mar, Calif.) as described (Jelinek and Weber, 1993).

Interaction of Small GTP-Binding Proteins With PI-3K in Vitro

Binding of ras, rac and rho to GST-PI3K was performed as described(Rodriguez-Viciana et al., 1995, 1996).

Antibodies, Immunoprecipitations and Immunoblotting

Monoclonal antibodies to bovine p85α (U1, U13), and p85β (T15) have beendescribed (End et al., Reif et al., 1993). A monoclonal antibody (I2)against bovine p85γ was developed in our laboratory. Rabbit polyclonalantiserum against GST-human p85α (AA 5-321) was kindly provided by Dr.P. Shepherd, University College London. Rabbit polyclonal antisera wereraised against a C-terminal peptide of p110δ (C)KVNWLAHNVSKDNRQ₁₀₄₄ andagainst an N-terminal peptide of human p110α (CGG)SVTQEAEEREEFFDETRR₈₈.To raise antibodies directed against the phosphorylated form of p110δ,the peptide sequence 1044 was phosphorylated at the serine residueduring peptide synthesis. An antiserum to the C-terminus of human p110α(KMDWIFHTIKQHALN) was kindly provided by Dr. Roya Hooshmand-Rad (LudwigInstitute for Cancer Research, Uppsala, Sweden). Antibodies wereaffinity-purified on peptides coupled to Actigel (SterogeneBioseparations, Arcadia, Calif.) or to AF-Amino ToyoPearl TSK gel (ToshoCo, Japan). Antibodies were found to be specific for the PI3K to whichthey were directed (tested against the following panel of PI-3K,expressed in Sf9 cells: bovine p110α, human p110β, (C. Panaretou and R.S.; unpublished results), human p110γ (Stoyanov et al., 1995), p110δ,PI-specific 3-kinase (Volinia et al., 1995). Peripheral blood cells werepurified over a ficoll gradient (Lymphoprep; Nycomed, Oslo, Norway).Neutrophil cytosol was prepared by sonication as described (Wientjes etal., 1993). Lysis buffer was 1% Triton-X100, 150 mM NaCl, 1 mM EDTA, 1mM NaF, 1 mM NaVO₃, 1 mM DTT, 1 mM PMSF, 0.27 TIU/ml aprotinin and 10 μMleupeptin. In some experiments, 1 mM disopropylfluorophosphate and 27 mMNa-p-tosyl-L-lysine chloromethyl ketone (hydrochloride) were added.Lysis buffer used for cytokine experiments was 50 mM Tris.HCl, pH 7.5,10% (v/v) glycerol, 1% (v/v) NP-40, 150 mM NaCl, 100 μM sodiummolybdate, 500 μM sodium fluoride, 100 μM sodium orthovanadate, 1 mMEDTA, 40 μg/ml PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.7 μg/mlpepstatin, 1 mM DIFP, 1 mM TLCK). Cytokine-stimulated cells werepelleted and lysed at 2×10⁷ cells/ml as described (Welham and Schrader,1992) with the exception that lysates were clarified for 5 min in amicrofuge ay 4° C. prior to further analyses. Immunoprecipitations werecarried out as described (Welham et al., 1994a) PDGF-receptor peptide(YpVPMLG) was coupled to Actigel according to the manufacturer'sinstructions. C-terminal antiserum to p110δ was used for bothimmunoprecipitations and immunoblotting. For p110α, the C- andN-terminal antisera were used for immunoprecipitations and Westerns blotanalysis, respectively.

SDS-PAGE and immunoblotting were carried out as described (Laemmli,1970; Welham and Schrader, 1992; Welham et al., 1994a). Antibodies wereused at the following concentrations for immunoblotting: 4G10,antiphosphotyrosine monoclonal antibody at 0.1 μg/ml; anti-p110α andp110δ at 0.25 μg/ml; anti-p85 at 1:4000; anti-c-kit (Santa CruzBiotechnology, sc-168) at 0.4 μg/ml, anti-SHP (Santa Cruz Biotechnology,sc-280) at 0.1 μg/ml and anti-IRS-2 (gift of Dr. M. White, JoslinDiabetes Center, Boston, Mass.) at 1:1000.

Both goat and anti-mouse and goat anti-rabbit horseradishperoxidase-conjugated antibodies (Dako, Denmark) were used at aconcentration of 0.05 μg/ml. Immunoblots were developed using the ECLsystem (Amersham). Blots were stripped and reprobed as previouslydescribed (Welham et al., 1994a).

Injection of CSF-1 Stimulated Mouse Macrophages With Antibodies to p110δand p110α

The murine macrophage cell-line, BAC1, was used in antibody microinjection experiments. The peptide polyclonal antibodies to p110δ weredirected to either the C-terminal peptide 1044, (described p17 Materialsand Methods), or to the peptide sequence (C)R222KKATVFRQPLVEQPED_(238.)Polyclonal sera were affinity purified before micro injection and wereused at a concentration of 0.5-5 mg/ml. A control peptide polyclonalantisera to human P110α is as described on p17 of Materials and Methods.Before micro injection, Bac1 cells were starved of Colony StimulatingFactor 1 (CSF1) for 24 hours. Antibodies were then injected into CSF1starved cells and exposed to CSF1 for 10-15 minutes before visualisationof the cytoskeleton of micro injected Bac1 cells with phalloidinconjugated rhodamine, (preparation and visualisation of cells is asdescribed in Allen et al 1997).

Cell Stimulations

Stimulation of cells with different growth factors was carried out asdescribed (Welham and Schrader, 1992) with the exception that cells wereresuspended at 2×10⁷/ml in serum-free RPMI prior to stimulations.Chemically synthesized murine IL3 and IL4 were kindly provided by Dr.Ian Clark-Lewis (University of British Columbia, Vancouver). Recombinantmurine SCF was purchased from R&D Systems Europe (Abingdon, Oxon). Theconcentration of growth factors and duration of stimulation (2 minutesfor SCF; 10 minutes for IL3 and IL4) had been previously optimised toobtain maximal levels of tyrosine phosphorylation of receptors andcellular substrates. These were as follows, IL3 at 10 μg/ml (Welham andSchrader, 1992), IL4 at 10 μg/ml (Welham et al., 1994a) and SCF 50 ng/ml(M. J. W., unpublished observations).

Northern Blot Analysis

Northern blots of human polyA+ RNA (Clontech) were hybridized withrandom prime-labelled EcoRI fragment II of pBluescript clone 0_(9.1).Stripping and reprobing using the following subsequent probes was thenperformed: internal EcoRI-XhoI 2.1 kb fragment from human p110α (Voliniaet al., 1994) and EcoRI-XhoI 5 kb cDNA of human p110β (C. Panaretou;unpublished results).

Using the above described materials and methods we were able toelucidate data which describes the novel lipid kinase and in particulara PI3 Kinase which we have termed p110δ. Data relating to this kinasewill now be described with a view to comparing p110δ with other membersof the PI3 Kinase group so as to compare and contrast their respectivecharacteristics.

Results

Cloning of p110δ

Degenerate primers based on conserved amino acid sequences (GDDLRQD andFHI/ADFG) in the kinase domain of bovine p110α and S. cerevisiae Vps34pwere used in RT-PCR reactions with mRNA from the human MOLT4 T cellleukaemia. A partial cDNA, homologous but different from other knownhuman PI3K, was obtained. This PCR fragment was used as a probe toscreen a U937 monocyte library, and to isolate the corresponding fulllength clone (for details, see Materials and Methods and FIG. 9).Sequence analysis revealed a potential open reading frame, preceded byan in-frame stop codon. The potential start codon was also found to liein a favourable context for translation initiation (Kozak, 1991). Thisopen reading frame of 3135 nucleotides predicts a protein of 1044 aminoacids with a calculated molecular mass of 119,471 daltons (FIG. 1A).Comparison of the amino acid sequence with other PI3K showed that thisprotein is most closely related to human p110β (58% overall identity; Huet al., 1993), and more distantly to human p110α (41% identity; Voliniaet al., 1994), human G-protein regulated p110γ (35% identity; Stoyanovet al., 1995) and the human vps34p analogue (28% identity; Volinia etal., 1995). The new PI3K described here will be further indicated asp110δ.

Dot plot comparison at high stringency (FIG. 1B) shows that p110α, β andδ are very homologous in the p85-binding region (AA 20-140 of p110α;Dhand et al., 1994) as well as in the C-terminal PI-kinase (PIK) domain(HR2) and catalytic core (AA 529-end of p110α, Zvelebil et al., 1996).An additional region of high sequence homology, spanning AA 370-470 ofp110δ, was found in between the p85 binding site and HR2. This regioncontains the so-called HR3 signature (WxxxLxxxlxIxDLPR/KxAxL) which isconserved in all p85-binding PI3Ks and in p110γ. The most N-terminalarea of sequence difference between p110α and p110β/δ overlaps with theregion defined in p100α as being sufficient for Ras binding (AA 133-314in p110α; Rodriguez-Viciana et al., 1996). Two additional structuralmotifs were identified in p110δ. The first is a proline-rich region(FIG. 1B, C) for which molecular modelling indicates that it can form aleft-handed, polyproline type-II helix with the potential to interactwith SH3 domains (data not shown). In the corresponding region, p110αand p110β lack crucial prolines to allow a similar fold. The secondmotif is a basic-region, leucine-zipper (bZIP)-like domain, immediatelyC-terminal of HR3 (FIG. 1B, C). A bZIP region is present in both p110δand p110β (and also in the Drosophila p110 (Leevers et al., 1997)),whereas the basic component of this domain is less prominent in p110α(FIG. 1C). Modelling of the p110δ ZIP region shows that its arrangementof L/V/I residues easily accommodates the formation of a helix structurewhich can form a coiled-coil dimeric protein zipper complex (data notshown).

p110δ Binds the p85 Adaptor and Ras Proteins

In order to verify the prediction from amino acid sequence comparisonthat p110δ might bind p85 subunits, p110δ was expressed in insect cellsas a glutathione-S-transferase (GST)-fusion protein, together withrecombinant baculoviruses encoding p85α, p85β or p85γ (the latter is a55 kDa bovine p85 isoform homologous to p55^(PIK), p55α and p85/AS53(Pons et al., 1995; Inukai et al., 1996; Antonetti et al., 1996)). As isclear from FIG. 2A all p85 adaptor subtypes efficiently co-purified withGST-p110δ from co-infected cells.

The question of whether different class I p110 catalytic subunits showbinding preference for different p85 adaptor proteins in vivo has notbeen previously addressed. Using antiserum specific for p110δ, we foundthat both p85α and p85β were present in p110δ immunoprecipitates fromdifferent white blood cells (FIG. 2B shows the data for humanneutrophils; note that p85γ is not expressed in leukocytes). Similarresults were obtained for p110α (data not shown). In these immunecomplexes, a 45 kDa protein reactive with p85α antibodies was alsoobserved (FIG. 2B). The nature of this protein is currently unclear, butit might be similar to a 45 kDa protein previously described to bepresent in p85 and p 110 IPs from various tissues (Pons et al., 1995).

P110α and p110β have been shown to interact with Ras-GTP (Kodaki et al.,1995; Rodriguez-Viciana et al., 1994 and 1996). The region required forthis interaction lies between AA 133 and 314 of these PI3Ks(Rodriguez-Viciana et al., 1996). Despite the relatively low sequenceconservation with p110α and p110β in this region (FIG. 1C), certainapparently critical amino acids are conserved as p110δ does interactwith Ras in vitro, in a GTP-dependent manner (FIG. 2C).

p110δ Binds Ras, But Not Rac or Rho

Incubation of GST-p110δ/p85α was found to retain GTP-bound wild-type rasor oncogenic V12-ras (FIG. 2C). This was not the case with GDP-loadedras, or with A38-ras, a functionally dead ras mutant. Similar as forp110α, no binding of rho and rac could be demonstrated (data not shown).

Lipid Kinase Activity of p110δ

When tested in the presence of Mg²⁺, p110δ was found to phosphorylatePtdIns, PtdIns4P and PtdIns(4,5)P₂ (FIG. 3A). HPLC analysis confirmedthat these lipids are phosphorylated at the D3 position (FIG. 3B).Substrate preference in vitro was PtdIns>PtdIns4P>PtdIns(4,5)P₂ (datanot shown). Lipid kinase activity was lower in the presence of Mn²⁺ thanin the presence of Mg²⁺ (tested over the concentration range of 0.25 to16 mM; data not shown). Specific activity of p110δ, isolated from Sf9cells, was a factor 2-5 lower than that of p110α (data not shown). Takentogether, these data establish p110δ as a genuine class I PI3K.

P110δ Does Not Phosphorylate p85 But Autophosphorylates.

The p85 subunit has been demonstrated to be a substrate for aMn²⁺-dependent phosphorylation by the p110α catalytic subunit (Carpenteret al., 1993; Dhand et al., 1994). In contrast, GST-p110δ failed tophosphorylate coexpressed p85α, p85β or p85γ under a variety of in vitroconditions (partial data shown in FIG. 4A; no activity was seen eitherin the presence of Mg²⁺ or Mn²⁺). p85γ lacks an SH3 domain, and theabsence of phosphorylation of this molecule by p110δ argues against thepossibility that an intermolecular interaction of the p85α/β SH3 domainwith the p110δ proline-rich region is locking up the p85 molecules forefficient phosphorylation by p110δ. In order to exclude that p110δ hadalready fully phosphorylated p85 during the in vivo co-expression ininsect cells, exogenous purified p85α was added to immobilizedGST-p110δ. After washing away the excess p85, bound p85 was found to beefficiently phosphorylated by p110α, but again not by p110δ (data notshown). When untagged p110δ, in complex with 85α or p85β, was subjectedto an in vitro kinase assay in the presence of Mn²⁻, p110δautophosphorylated ((FIG. 4B note that this activity is largely absentin immobilised GST-p110δ (FIG. 4B)). Such phosphorylation was not seenin p110α/p85 complexes, in which again p85 was found to bephosphorylated (FIG. 4B). Phosphoamino acid analysis showed that thephosphorylation on p110δ occurred on serine (FIG. 4B). Both thephosphorylation of p85 by p110α and the autophosphorylation of p110δwere observed to be largely Mn²⁺-dependent, with only very weakphosphorylation in the presence of Mg²⁺ (data not shown).Autophosphorylation of p110δ resulted in reduced lipid kinase activity.

In order to exclude the possiblity that the observed phosphorylation ofp110δ was due to a coprecipitated protein kinase, a kinase-defectivep110δ mutant was generated. This was done by converting arginine 894 toproline in p110δ, generating p110δ-R894P. The mutated arginine residueis located in the conserved DRX₃NX₁₂₋₁₃DFG motif of the kinase domain,likely to be part of the catalytic loop as in protein kinases (Taylor etal., 1992, Zvelebil et al., 1996). A similar mutation in bovine p110α(R916P) has been found to completely knock out catalytic activity (Dhandet al., 1994). As is clear from FIG. 4C, p110δ-R894P, expressed ininsect cells, was no longer phosphorylated in precipitates of p110δ,indicating that the latter has indeed autophosphorylation capacity.Likewise, lipid kinase activity was found to be lost by p110δ-R894P(data not shown).

We have produced polygonal antisera to the phosphorylated form of p110δ.The C-terminal peptide sequence 1044 was phosphorylated at the serineresidue 1033 and used to immunize rabbits. The antisera directed againstthe phosphorylated peptide has enabled us to establish that p110δ isphosphorylated in vivo and upon cytokine stimulation thisphosphorylation is enhanced (results not shown).

Drug Sensitivity of p110δ Catalytic Activity

p110α and δ lipid kinase activity were found to exhibit a similarsensitivity to inhibition by wortmannin and LY294002 (FIG. 5), with anIC₅₀ of 5 nM (for wortmannin) and 0.5 μM (for LY294002). Likewise, theautophosphorylation activity of p110δ was also inhibited by wortmanninin the nanomalar range (data not shown)

Tissue Distribution of p110δ

The expression pattern of p110δ was investigated by Northern blotanalysis of polyA⁺ RNA of human tissues, and compared with that of p110αand p110β. A single messenger mRNA species of approximately 6 kb wasfound to be particularly highly expressed in white blood cellpopulations i.e. spleen, thymus and especially peripheral bloodleucocytes (the latter contains all white blood cells with only themajority of the erythrocytes being removed) (FIG. 6). In some Northernblot experiments, an additional −5 kb messenger for p110δ was alsoobserved (data not shown). Low levels of p110δ messenger RNA expressionwere found in most other tissues examined, although it is difficult toexclude the possibility that blood cell contamination is responsible forthis p110δ mRNA signal. p110α and p110β were also found to be expressedin most tissues examined (FIG. 6).

Antibodies specific for p110α and δ were then used to assay theexpression of these PI3K at the protein level. Upon testing differentrat tissues, a 110 kDa protein reactive with p110δ antibodies was foundin spleen and thymus, but not in any of the other tissues tested (FIG.7). This pattern largely confirms the data of the Northern blot analysisdescribed above. p110δ was also found to be present in both primary andtransformed white blood cells, independent of their differentiationstage (FIG. 7). In the primary blood cells, both the lymphoid andmyeloid cell populations were positive for p110δ whereas platelets werenot (FIG. 7). Both T (e.g. Jurkat, HPB All) and B (e.g. Raji, HFB1) celllines expressed p110δ (FIG. 7). The 110 kDa p110δ was not found inRat-1, NIH 3T3 and Swiss 3T3 fibroblasts, LS174T and COLO 320HSR colonadenocarcinomas, A431 epidermoid carcinoma, ECC-1 endometrial carcinomaand HEp-2 larynx carcinoma (FIG. 7) nor in CHO chinese hamster ovary,POC small-cell lung cancer cell line, porcine and bovine aorticendothelial cells, MDA-MB-468 breast adenocarcinoma, and primary humanmuscle and fibroblasts (data not shown). In conclusion, it appears thatp110δ is selectively expressed in leukocytes.

In contrast to p110δ, p110α was found in most of the tissues and celllines investigated, including the white blood cells (FIG. 7).

Micro Injection of Anti p110δ Polyclonal Antibodies Into CSF-1Stimulated Murine Macrophages

The possible function of p110δ was investigated further by a series ofmicro injection experiments of the murine macrophage cell-line, Bac1with antisera to p110δ and p110α. Prior to micro injection, Bac1 cellswere deprived of CSF1 for 24 hours. CSF1 deprivation primes cells todivide and become motile when subsequently exposed to CSF1. Affinitypurified anti p110δ polyclonal antibodies were micro injected into CSF1deprived Bac1 cells followed by exposure to CSF1 for 10-15 minutes.

The micro injected Bac1 cells show marked alterations in cellularmorphology. The normal cell membrane ruffling disappears and cytoplasmicretraction occurs. The cytoskeleton of micro injected Bac1 cells wasvisualised using a phalloidin-rhodamine conjugate and FIG. 10 shows arepresentative sample of such cells showing a disorganised cytoskeletalarrangement. The injection of anti p110α does not produce an equivalenteffect.

Interestingly a similar phenotype is shown by expression of thedominant-negative small GTP-binding protein rac, N17RAC. This suggeststhat p110δ may be part of the same signalling cascade that may beinvolved in cytoskeletal organisation and cellular motility.

p110δ is Involved in Cytokine Signalling

In leucocytes, p85-binding PI3Ks have been implicated in a wide varietyof signalling events including signalling via cytokine and complementreceptors, integrins, Fc receptors, B and T cell antigen receptors andtheir accessory molecules such as CD28 (reviewed by Stephens et al.,1993; Fry, 1994). Therefore, it is clear that a multitude of signallingprocesses could be potentially linked to p110δ. A crucial question iswhether selective coupling of p110δ to the above-mentionedsignalling/receptor complexes occurs in cells that also contain otherclass I PI3K, given the observation that different p110s seem to becomplexed with the same p85 isoforms (FIG. 2B). We addressed thisimportant question in the context of cytokine signal transduction,operative in diverse types of leukocytes.

Different families of cytokines transduce signals via discrete classesof receptors that share common gp130, β or γ chains, or via receptorswith intrinsic tyrosine kinase activity (reviewed in Taga and Kishimoto,1995). Whereas PI3K activation by cytokines signalling via gp130 has notbeen reported, activation of p85-binding PI3K in response to cytokinesignalling via the common β chain (eg IL3), common γ chain (eg IL4), orvia tyrosine kinase receptors (such as c-kit, which binds Stem CellFactor (SCF)) has been demonstrated (Wang et al, 1992; Gold et al,1994). We examined the ability of IL3, IL4 and SCF to couple to p110δand p110α in cytokine-dependent leukocyte cell lines. An identicalpattern of phosphotyrosine-containing proteins, specific to the cytokineused for stimulation, was found to co-precipitate with p110α and p110δantibodies (FIG. 8, panel a). In the IL3- and IL4-responsive Ba/F3 pre-Band myeloid progenitor FD-6 cell lines (FIG. 8A; data for FD-6 are notshown), IL3-treatment induced the appearance in p110α/δ IPs of anunknown protein of 100 kDa and the 70 kDa protein tyrosine phosphatase,SHP2 (FIG. 8A, panel b). The 170 kDa protein co-precipitated upon IL4stimulation (FIG. 8A, panel a) was shown by immunoblotting to be IRS-2,the major substrate of IL4-induced phosphorylation in these cells (datanot shown). FIG. 8B shows the results of similar analyses in MC/9 mastcells. Following SCF stimulation, both p110α and p110δ IPs contained anunidentified 100 kDa tyrosine-phosphorylated protein as well as a 150kDa protein identified as c-kit, the SCF receptor (FIG. 8B, panels a andb). Taken together, these data indicate that p110α and p110δ show noapparent differences in their recruitment to a variety of activatedcytokine receptor complexes. In addition, the implication in cytokinesignalling of at least two members of the p85-binding PI3K class revealsa previously unrecognised complication of signal transduction pathwaysdownstream of these cytokine receptors.

Expression of PI3 Kinase p110 Sub Units in Murine and Human MelanomaCell-Lines.

The expression of p110δ was further investigated in various murine andhuman melanoma cell-lines. A characteristic feature of a melanoma is theaggressive nature of the metastasis associated with this cancer. Thepossible involvement of p110δ in metastasis was investigated byanalysing the relative abundance of p110δ protein in a range of murineand human cell-lines. Western blots were used to assess the levels ofp110α and β as well as p110δ. J774, a murine cell-line, was used as apositive control for the murine western blots. Neonatal melanocytes wereused as a control for the human western blot. Table 1 indicates thatp110α and β are constitutively expressed in both control and melanomacell-lines of both murine and human origin. Interestingly, the murinecontrol cell-line J744 shows markedly reduced levels of p110δ whencompared to the murine melanoma cell-lines. However detectable levels ofp110δ are found in human neonatal melanocytes. This may be explained bythe nature of these human control cells. The expression of p110δ inthese control cells may be explained by the relatively recent migrationof these cells in the human skin and therefore residual levels of p110δmay be present in these cells. Adult melanocytes have prolongedresidence in skin and the level of p110δ may be reduced to undetectablelevels commensurate with their terminal differentiation.

We have described a novel human p110 subunit, p110δ, which is part ofthe PI3 kinase family. p110δ shows a restricted expression pattern, onlyaccumulating to significant levels in white blood cells populations andparticularly in peripheral blood leucocytes. The motile nature of thesecells has lead us to propose that this member of the PI3 kinase familymay be involved in regulating the motility of cells via cytoskeletalreorganisation. The data relating, to murine and human melanoma celllines is interesting but inconclusive with regard to human melanomas.The use of tissue biopsies of normal human melanocytes and humanmelanomas will allow this to be resolved. TABLE 1 Expression of p110Subunits in Murine Melanomas Cell-line Characteristic δ α β ReferenceMurine J774 Control − + + This study Melan-c Melanoma − + + Melan-plMelanoma − + + Wilson et al 1989 Melan-a Melanoma − + + Wilson et al1989 Tu-2d Mel-ab Melanoma +/− + + Dooley et al 1988 Mel-ab-LTR-Melanoma + + + Dooley et al 1988 Ras2 Mel-ab-LTR Melanoma + + + Dooleyet al 1988 Ras 3 Mel-ab-pMT Melanoma + + + Dooley et al 1988 B16 F1Melanoma + + + Fidler et al 1975 (weakly metastatic) B16 F10Melanoma + + + Fidler et al 1975 (highly metastatic) Human A375PMelanoma − + + Easty et al 1995 (weakly metastatic) A375M Melanoma + + +Easty et al 1995 (highly metastatic) WM164 Melanoma + + + Easty et al1995 WM451 Melanoma + + + Easty et al 1995 DX3 Melanoma + + + Ormerod etal 1986 (weakly metastatic) DX3-LT5.1 Melanoma − + + Ormerod et al 1986(Highly metastatic) Control Primary cells + + + This study (humanneonatal melanocytes)

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1-29. (Canceled).
 30. A method for identifying an agent which modulatesactivity of a p110δ molecule, comprising exposing said p110δ molecule tosaid agent, observing activity of said p110δ molecule in the presence ofsaid agent, said activity selected from the group consisting of kinaseactivity, autophosphorylation and cell mobility, and comparing saidactivity to activity of said p110δ molecule in the absence of saidagent, any difference therebetween indicating that said agent modulatesactivity of p110δ, wherein the amino acid sequence is set forth as SEQID NO:
 1. 31. The method of claim 30, wherein said activity is kinaseactivity.
 32. The method of claim 30, wherein said activity isautophosphorylation.
 33. The method of claim 30, wherein said activityof p110δ is cell mobility.
 34. The method of claim 30, comprisingcarrying out said method in vitro.
 35. The method of claim 30,comprising exposing a cell which expresses said p110δ to said agent. 36.The method of claim 35, wherein said cell has been transfected by anisolated nucleic acid molecule which encodes said p110δ.
 37. The methodof claim 30, comprising exposing said p110δ to said agent and to p85.38. The method of claim 35, wherein said cell is an insect cell or amammalian cell.
 39. The method of claim 30, wherein said agent is anantagonist.